Sphingolipids (SLs) play roles in a wide variety of cell functions, including cell-cell interactions, cell growth and differentiation, and signal transduction. They can interact with cholesterol to form membrane domains, and data from many studies suggest that the plasma membrane (PM) SL and cholesterol composition is tightly regulated. Studies in this renewal application will focus on how SLs regulate transport of certain lipids and proteins to and from the cell surface. Using human skin fibroblasts, we will pursue three Specific Aims to test our central hypothesis that cellular SLs are critical for regulating these transport pathways. Under Aim 1, we will test the hypothesis that SLs regulate different mechanisms of clathrin-independent endocytosis by their ability to promote PM domain formation and to organize endocytic cargo for subsequent internalization. The effect of a novel SL analog on PM domain formation and organization of endocytic cargo will also be examined in light of our recent discovery that this SL selectively inhibits caveolar endocytosis. Under Aim 2, we will test the hypothesis that glycosphingolipids regulate caveolar endocytosis by inducing the clustering of integrins and other transmembrane proteins into PM domains, resulting in their activation and transmembrane signaling. The effects of the inhibitory SL analog on these processes will also be studied, and we will examine other cell systems in which inhibition of integrin function can have a profound effect on cell function. We will also study the regulation of RhoA- and Cdc42-dependent endocytosis by SLs and test our working model that this regulation results from sphingomyelin's ability to modulate the translocation of these GTPases, and perhaps other molecules, to the PM. Under Aim 3, we will investigate the possibility that transport of selected lipids and proteins along the secretory pathway may be specifically linked to endocytosis via caveolae. These studies will also test potential mechanisms by which inhibition of SL and isoprenoid synthesis block secretory transport of selected proteins, and the potential role for lipids in regulating some of the machinery involved in this secretory pathway. Data resulting from the proposed studies will lead to a greater understanding of the regulation of endocytosis and secretion and may lead to techniques for blocking uptake of certain toxins or pathogens that enter cells by clathrin-independent internalization mechanisms. Studies with the inhibitory lipid may also have important future applications in other cell systems where inhibition of integrin function can have significant effects on inflammatory responses, or cell migration and invasiveness.